Synthesis, characterization and targeted cell imaging applications of poly(p-phenylene)s with amino and poly(ethylene glycol) substituents

Article abstract of DOI:10.1039/c5ra08893c

A novel approach for bioconjugation associated with a fluorescent conjugated polymer is demonstrated. For this purpose, a conjugated polymer, poly(p-phenylene) (PPP), with lateral substituents, namely primary amino groups and poly(ethylene glycol) (PEG) chains, as a potential building block for polymer bioconjugates was synthesized and characterized. The synthesis was achieved through Suzuki polycondensation reaction in the presence of Pd(PPh₃)₄ catalyst by using independently prepared PEG and amino functionalized dibromo benzenes in conjunction with benzene diboronic acid. For the evaluation of the bioactive PPP labeled with folic acid (FA) as a potential targeted cell imaging probe, HeLa and A549 cancer cells were used. Cytotoxicity assay showed that the polymer was not toxic to either of the cells. Additionally, the fluorescence images showed that, depending on the level of the FA receptors on the cell surfaces, the fluorescent intensity in HeLa cells was obviously higher than A549 cells when treated with FA conjugated PPP-NH₂-g-PEG polymer. The resulting FA/PPP-NH₂-g-PEG conjugate was successfully used as a bioconjugate for targeting and specifically imaging FA receptor positive HeLa human cervical cancer cells.

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Synthesis, Characterization and Targeted Cell Imaging
Applications of Poly(p-phenylene)s with amino and poly(ethylene
glycol) Substituents
Received 00th January 20xx,
Accepted 00th January 20xx
Huseyin Akbulut,a Bahar Guler,b Suna Timurb* and Yusuf Yagcia,c*
DOI: 10.1039/x0xx00000x
A novel approach for bioconjugation associated with fluorescent conjugated polymer is demonstrated. For this purpose, a
conjugated polymer, poly(p-phenylene) (PPP), with lateral substituents, namely primary amino groups and poly(ethylene
glycol) (PEG) chains, as potential building block for polymer bioconjugates was synthesized and characterized. The
synthesis was achieved through Suzuki polycondensation reaction in the presence of Pd(PPh3)4 catalyst by using
independently prepared PEG and amino functional dibromo benzenes in conjunction with benzene diboronic acid. For the
evaluation of the bioactive PPP labeled with folic acid (FA) as a potential targeted cell imaging probe, HeLa and A549
cancer cells were used. Cytotoxicity assay showed that the polymer was not toxic to the both cells. Additionally, the
fluorescence images showed that, depending on the level of the FA receptors on the cell surfaces, the fluorescent intensity
in HeLa cells was obviously higher than A549 cells when treated with FA conjugated PPP-NH2-g-PEG polymer. The resulting
FA/PPP-NH2-g-PEG conjugate was successfully used as bioconjugate for targeting and specifically imaging of FA receptor
positive HeLa human cervices cancer cells.
www.rsc.org/
light-harvesting systems, bio-sensors, photonics and nano-
electronic devices.6-8
Introduction
To generate a bioconjugation system with synthetic polymer
Bio-functionalization of polymers of synthetic origin have
played a challenging role over the past decades in the wide
range of bioconjugation applications including the covalently
incorporation of synthetic polymers with biological materials
such as antibodies, peptides, proteins, enzymes,
carbohydrates, viruses or cells.1 Conceptually, this
complementary strategies over conjugation of peptides,
proteins or enzymes with synthetic polymers to design the
versatile molecular biomaterials, had been first considered as
and biomaterial through covalent linkage, the most common
method is the carbodiimide-mediated coupling reaction. As
cross-linking agents, carbodiimides (EDC or DCC)/N-
hydroxysuccinimide (NHS) gives coupling reaction between the
primary amine groups in polymer and the carboxyl groups in
biomaterial or vice versa positions. Other similar approaches
based on chemical modification of primary amine groups to
make them appealing different bioconjugation pathways.9-11
Recent applications related to amino-based systems include, in
situ enzymatic photo-patterning for manipulation of cell,12
amine-modified carbon nanotubes and nonporous carbon in
essential topic in pharmaceutical chemistry for
a few
decades.2-5 In line with recent discoveries of nanotechnology
and biotechnology, polymer bioconjugates have gone far the
field of biomaterial and gained enormous attraction in
material science consisting of artificial enzymes, biometrics,
controlled drug
releasing,13-14 non-natural amino acid-
mediated protein and polysaccharide conjugation for synthetic
biology purposes,15 bio-based covering material for surface
design and biosensor fabrication.16-17 Amino-rich polymer-
based nanoparticles for imaging-guided drug delivery and
therapy,18 biological labelling application of protein by
aminated luminescent nanoparticles,19 polyamine conjugates
in systematic in vivo short interfering RNA delivery
therapeutics,20 surface initiated living polymerization of N-
carboxyanhydride (NCA) to obtain polypeptides by grafting21-23
are the other typical uses and numerous other studies remain.
Although the existence of amine group on polymer provides a
unique property and advantage in bio-functionalization
process, water-solubility is essential additional requirement in
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biological application. Water solubility can be achieved by imaging of FA receptor positive HeLa human cervices cancer
water soluble supports such as poly(ethylene glycol) (PEG), cells. The FA conjugated PPP-NH2-g-PEG polymer was obtained
which comes to the forefront from a highly versatile class of by a simple bioconjugation method called EDC/NHS chemistry.
polyethers. PEG was also approved by the US-American Food The resulted conjugates were characterized by several
and Drug Administration for the consuming in human body, methods such as FTIR and fluorescence spectroscopy. Finally,
exhibiting no antigenicity, immunogenicity, or toxicity.24-33
the cellular internalization of fluorescent-based polymer was
Besides the effective role of PEG in bio-applications, advances visualized by fluorescence microscopy.
in the field of bio-imaging have profoundly impacted the use of
water soluble bio-polymers and concerns significantly focused
Results and discussion
on the fabrication of fluorescence-based organic conjugated
polymers relying on π-electron delocalization features.34
Among the vast number of conjugated polymers, PPP is the
most promising polymer in terms of relatively high
photoluminescence (PL), electroluminescence (EL) quantum
efficiencies and thermos-oxidative stability. PPP can be easily
functionalized to form a wide range of its derivatives due to
fact that in the synthesis processes, generally the monomer is
chosen as phenyl compounds allowing to many chemical
modification readily.35-40 Unfortunately, the main drawback,
PPP has, is the insolubility in many solvents, which limits their
processability. However, the addition of conformationally
appropriate alkyl or water soluble pendants to backbone can
lead to obtain soluble PPPs with sufficiently high molecular
weight.41-49
The instruction of bioactive polymer embodying amino and
PEG lateral substituents on the same linear conjugated chain
needs two different functional monomers in the time that
Suzuki polycondensation was chosen as the polymerization
technique. Suzuki coupling reaction providing more
straightforward access to obtain polymer with changeable
sequence ratio between amino functional and PEG monomers
was used to adjust the higher contribution of amino monomer
than PEG macromonomer to PPP structure. Amino
functionality is the crucial part of this study for the utilization
of the polymer for bioconjugation. By this association, the
preparation of primary amine functional monomer via Gabriel
synthesis is based on the transformation of the primary alkyl
halides into the primary amines.53-60 To obtain primary alkyl
halide, starting material was chosen as 2,5-dibromotoluene as
it can readily react with N-bromosuccinimide (NBS) to afford
allylic bromination of methyl group of toluene in the presence
of peroxide. In the first stage, the reaction between 1 and
conjugate base of phtalimide gives the corresponding N-alkyl
phtalimide. The efficiency of the reaction was increased by
using preformed potassium salt of phtalimide instead of
phtalimide. Reaction proceeded with a polar protic solvent
DMF under homogenous condition and the substitution
product methyldibromophenyl phenothiazine (2) was obtained
in a higher state of purity and in a shorter period of time.
Second stage, named as hydrazinolysis, is the treatment of
imide with hydrazine in methanol, results phthalhydrazide and
DBB-MA through intramolecular substitution. With the column
chromatograph, the amino compound purified and eliminated
from the side product of phthalhyrazide (Scheme 1).
Nowadays, the goal and the content of the studies in
biomedical field are focused on increasing delivery of the
variety
of
drugs,
gene
therapy
agents,
and
radiopharmaceuticals to the target tissue. A variety of ligands
has been examined for the development of efficient targeted
imaging, drug delivery and tracking systems.50 The folate
receptor has been proven as a highly selective overexpressed
tumor marker in epithelial lineage tumours such as ovarian,
colorectal, and breast cancer by the side of healthy tissue.51
Besides, it is well-known that folic acid (FA) has a high affinity
to the folate receptors and is widely used as a targeting ligand
for tumor-specific delivery. The inclusion of FA to the targeting
system simplifies the internalization of the molecules by the
receptor overexpressed cancer cells via receptor-mediated
endocytosis.52
In this study, we describe design and synthesis of fluorescent
conjugated polymer possessing amino and PEG side groups
(PPP-NH2-g-PEG) suitable for cell-targeting and imaging
studies. In this connection, it should be pointed out that there
exist several other promising approaches for surface
modification or targeting tissues of interest utilizing acid
functionalized polymers or anionic polysaccharides-based
nanoparticlesref. However, the benefits of our system are
several folds. In our approach, both amine and hydrophilic PEG
functionalities are attached using the same chemistry that
facilitates bio-functionality and water solubility, respectively.
The obtained polymers have excellent photophysical
properties that can be benefited for the image proposes
without any additional fluorophore. Besides, the some
problems associated with the nanoparticles are eliminated due
to the fully organic and biocompatible polymeric structure.
As will be shown below, the obtained water-soluble polymer
was decorated with FA for the targeting and specifically
Br
Br
CH3
O
NBS
CCl4
Br
Br
Br
KN
O
1
DMF
O
NH2
N
NH2NH2
Br
Br
Br
Br
O
DBB-MA
2
Scheme 1. Synthesis of amino functional monomer, DBB-MA.
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DBB-MA was characterized by 1H-NMR analysis. Singlet peak at components in the conjugated polymer. In our work, we
1.59 ppm was assigned to -NH2 protons. To further confirm the deliberately used the molar ratio of DBB-PEG/DBB-MA as 1/3
existence of primary amine, hydrogen–deuterium exchange to achieve sufficient chain growth since DBB-PEG is relatively
reaction was performed. Notably, after the deuterium less reactive due to the polymeric and hydrophilic nature.
exchange with D2O, this peak disappears. The detailed Moreover, once adequate hydrophilicity is achieved, the
assignment of the all characteristic peaks is presented in amino functionality is crucial for the subsequent
1
bioconjugation. In the H-NMR spectrum, characteristic benzyl
Figure S1 (Supplementary Information).
The other dibromo benzene component for the subsequent amine protons appear at 1.96 ppm as a broad peak which
Suzuki coupling, water soluble macromonomer was obtained shifts to 4.71 ppm after D2O exchange (Figure 2S,
by mild Steglich method with the favorable catalytic action of Supplementary Information).
4-dimethylaminopyridine (DMAP).61 Esterification reaction The Mn and Mw/Mn values of PPP-NH2-g-PEG soluble polymer
between PEG monomethylether and 2,5-dibromobenzoic acid were found to be 24500 g mol-1 and 1.41 as determined by gel
under the standard condition gave the desired product in good permeation chromatography (GPC). This value should be taken
yield (Scheme 2). After the elimination of the excess amount of as the minimum estimation63-64 since the polymer has
dicyclohexylcarbodiimide (DCC) and unreacted PEG hydrophilic side chain and comb-like structure and the GPC
monomethylether by column chromatography, the system was calibrated with linear polystyrene standards.
macromonomer with Mn = 5550 g mol-1 and Mw/Mn = 1.24 was Figure 1 shows the GPC traces of the macromonomer and the
obtained.
final conjugated polymer. The trace of the conjugated polymer
consists a weak shoulder at high elution volume pertaining to
the unreacted PEG macromonomer. This may arise from the
difficulty to achieve exact equimolarity particularly when
Suzuki coupling components with different molecular weights
are used. Due to the similar solubility properties, the
separation of the macromonomer by extraction was not
possible. However, the presence of small but some
macromonomer in the conjugated polymer would not affect
the ultimate use in the bioconjugation process, since PEG is
known to be a biocompatible polymer.
Scheme 2. Synthesis of water soluble macromonomer, DBB-PEG, via Steglich
esterification.
Synthesis of PPP with primary amine groups and PEG side chains.
The combination of PEG and amine functional monomer on
the PPP backbone was achieved by Suzuki condensation
polymerization using 1,4-benzenediboronic acid as the
coupling partner in the presence of palladium catalyst (Scheme
3).
O
O
n
NH2
Br
O
Br
Br
Br
DBB-MA
DBB-PEG
Pd(PPh3)4
THF / H2O
K2CO3
(HO)2B
B(OH)2
O
O
n
Figure 1. GPC traces of PPP-NH2-g-PEG and DBB-PEG.
NH2
O
UV-Vis absorption spectra of the components of the coupling
polymerization and the final polymer are shown in Figure 2.
Expected strong absorption band of PPP-NH2-g-PEG was due to
the presence of supplementary conjugated phenylene rings in
the main chain that can be regarded as the further evidence
for the formation of PPP backbone.
p
m
PPP-NH2-g-PEG
Scheme 3. Synthesis of PPP-NH2-g-PEG via Suzuki condensation polymerization.
Obviously, if desired, the polymer may further be modified by
the same reaction through terminal boronic acid or
bromobenzene groups.61-62 Addition of coupling partner makes
it possible to arrange the sequence proportion of the
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a maximum emission at 410 nm. Upon conjugation, the
fluorescence profile of the polymer retained without any
major changes. However, conjugation step caused a significant
decrease in the fluorescence intensity. However, the remained
fluorescence signal is still high enough to use the system for in
vitro cell imaging experiments, which cannot be realized by
conventional fluorophores.
Figure 2. UV absorption spectra of PPP-NH2-g-PEG, DBB-PEG and DBB-MA in DMF,
solution (0.1 g/L).
The excitation and fluorescence emission spectra of PPP-NH2-
g-PEG in DMF and water were recorded (Figure S3,
Supplementary Information). As can be seen, while the
excitations in both solvents present similar trend, the
corresponding emission in water is slightly blue shifted relative
to that in DMF. Moreover, the aggregations that are taking
place during the nanoparticles formation through self-
assembling of PPP backbones in solution create the red shifted
shoulders in asymmetric shape.65-66
Figure 3. Fluorescence spectra of PPP-NH2-g-PEG and FA/PPP-NH2-g-PEG polymer
conjugates.
FTIR analysis was used to confirm the incorporation of the FA
in the PPP-NH2-g-PEG, which is shown in Figure S4 (in
Supplementary Information). FA is formed from a pteridine
ring, p-amino benzoic acid and glutamic acid residue. As can be
seen from the FTIR analysis of pure FA, the most characteristic
peak absorption which was attributed to -COOH and carbonyl
groups (C=O) and aromatic C=C structures on both phenyl and
pteridine rings was clearly demonstrated itself at the 1684 and
1606 cm-1. After the conjugation of FA with PPP-NH2-g-PEG
(which will be then called as FA/PPP-NH2-g-PEG), the
absorption peaks at 1684 and 1611 cm-1 may be belong to the
C=O stretching which is related to amide-I bond. Furthermore,
the absorption band between 3300 and 3600 may have
belonged to hydroxyl stretching bands that comes from FA.
Normally, as in the case of most conjugated polymer, PPP
derivatives have a rigid structure which causes the insolubility
problem in common organic solvents. The grafting of relatively
long and flexible side chains to the polymer backbone has
been a major approach to deal with this problem. On the other
hand, the side chains features may change the optical and
electronic properties of PPP backbone.34 In our approach, the
solubility is achieved without considerable negative effect on
the fluorescence properties of PPP backbone.
Bioconjugation of the FA to PPP-NH2-g-PEG.
The polymer and obtained bioconjugate were characterized by
various spectroscopic and microscopic investigations. PPP-NH2-
g-PEG was conjugated with FA via a chemical method outlined
in Scheme 4.
Cell culture
Cytotoxicity assay. Cytotoxic effects of PPP-NH2-g-PEG and
FA/PPP-NH2-g-PEG polymer at different concentrations were
examined by MTT assay on A549 and HeLa cells. As
demonstrated in the Figure 5, the MTT assay results for HeLa
and A549 cells, toxicity of the FA/PPP-NH2-g-PEG polymer was
slightly higher than non-conjugated polymer on HeLa cell lines,
while both of them showed similar toxicity on A549 cell lines.
The differences between toxicity of FA conjugated and non-
conjugated polymer may depend on the cellular uptake of the
samples. Recently, the expression levels of FA receptors on the
surfaces of both cell lines were investigated by using both
genomic and proteomic approaches, PCR and flow cytometry
analysis, and were found to be overexpressed on surface of
HeLa cells compared to A549 cell lines.67 It is attributed that
Scheme 4. Schematic illustration of the bioconjugation.
Fluorescence properties of PPP-NH2-g-PEG polymer before and
after bioconjugation were evaluated. As can be seen from the
fluorescence spectra illustrated in Figure 3, PPP-NH2-g-PEG has
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the increase in cytotoxicity was caused by enhanced cellular receptor poor A549 cells, no differences were observed
uptake of FA conjugated polymer in FA receptor positive HeLa between the fluorescence signal both of FA conjugated and
cells via receptor-mediated endocytosis.
non-conjugated polymer samples (Figure 6A-B). As it can be
seen from the fluorescence images, the fluorescent intensity in
HeLa cells was obviously higher than A549 cells, depending on
the uptake selectivity of FA conjugated polymer. Also,
fluorescence intensity of the FA/PPP-NH2-g-PEG polymer in FA
pre-treated HeLa cells significantly decreased because of the
Figure 5. The effect of PPP-NH2-g-PEG and FA/PPP-NH2-g-PEG polymer on cell viability
of HeLa cells (A) and A549 cells (B). Values are the mean ± standard deviation (n = 6).
Fluorescence microscope images. FA receptor positive cells have
no pores or channels for the diffusion of the polymeric
materials into the cells. In order to achieve more efficient
therapy and imaging studies, it is important to use of
membrane permeable agents.68 FA functionalized materials
constitute a considerable target for tumor specific delivery of
Figure 6. Fluorescence microscopy imaging of HeLa and A549 cells. Cells
were treated with FA/PPP-NH2-g-PEG polymer directly and after being pre-
treated with free FA (A). Cells were treated with non-conjugated PPP-NH2-g-
PEG polymer (B) for 2 h at 37oC, overlap of two images, control nuclei
staining with DAPI.
variety
of
drugs,
gene
therapy
agents,
and
radiopharmaceuticals for FA receptor rich cell lines such as
HeLa.69 For the investigation of FA/PPP-NH2-g-PEG conjugate
as a potential targeted cell imaging probe, fluorescence
microscope images of the HeLa and A549 cells were taken
after incubation of the samples. The fluorescence images of
HeLa and A549 cells treated with the FA/PPP-NH2-g-PEG
polymer and non-conjugated polymer were given in Figure 6.
According to fluorescence microscope images of cells, the
visual fluorescence signal is increased in HeLa cells after being
treated with FA/PPP-NH2-g-PEG polymer in comparison with
non-conjugated polymer. As shown in Figure 6-A,
functionalization of the PPP-NH2-g-PEG polymer with FA
increased the cellular uptake in HeLa cells through FA
receptors on cell surfaces while the non-conjugated polymer
was not endocytosed (Figure 6-B). In contrast to this, for FA
saturation of FA receptors with free FA. Based on these results
it can be said that the internalizing of the polymer by the HeLa
cells is dependent on endocytosis via FA receptors. Besides, all
of this data is coherent with the cytotoxicity assay results.
Experimantal
Materials
Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), 2,5-
Dibromo-benzoic acid, 1,4-benzene-diboronic acid, N-
Bromosuccinimide (NBS), 4-(N,N'-dimethyl) amino pyridine
(DMAP), dicyclohexylcarbodiimide (DCC), poly(ethylene glycol)
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mono methyl ether (PEG2000), Phthalimide potassium salt, Synthesis of (2,5-dibromophenyl)methanamine (DBB-MA).
hydrazine monohydrate (98%) and Benzoyl peroxide, Folic The solution of (0.381 g, 0.95 mmol) in 25 mL absolute
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide ethanol with 3.0 mL hydrazine were stirred at 90 °C for 5 h.
2
acid,
hydrochloride,
Sigma
N-Hydroxysuccinimide,
tetrazolium
3-(4,5- After allowing cooling to room temperature, the mixture was
bromide, extracted with CH2Cl2 and 10% NaHCO3. The organic layer was
dimethylthiazol-2-yl)-2,5-diphenyl
sodium dodecyl sulfate, Diamino-2-phenylindol were dried over Na2SO4 and reduced in vacuo. The residue was
purchased from Sigma-Aldrich and used without any further purified by column chromatography (Al2O3 (neutral), THF) to
1
purification. All solvents were purified and dried. Water was yield (DBB-MA) (145 mg, 57%, yellow oil). ). H-NMR (CDCl3,
purified in a Milli-Q plus System (Milli-pore).
500 MHz): δ 1.59 (br, 2H), 3.87 (s, 2H), 7.23 (dd, J = 8.4, 2.4 Hz,
1H), 7.38 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H).
Measurements
1H-NMR spectra were performed with an Agilent VNMRS 500
MHz, and chemical shifts were recorded in ppm units using
tetramethylsilane as an internal standard. UV/vis absorbance
measurements were recorded on a Shimadzu UV-1601
spectrometer. Fluorescence measurements were performed
with a model LS-50 spectrometer from Perkin Elmer at room
Synthesis of PEG macromonomer (DBB-PEG). 2,5-Dibromo-
benzoic acid (4.0 g, 14.3 mmol), DMAP (174.7 mg, 1.43 mmol),
Me-PEG2000-OH (25.74 g, 12.87 mmol) were dissolved in 80 ml
of dry CH2Cl2 under nitrogen atmosphere. To this solution was
added DCC (3.246 g, 15.7 mmol) in 20 mL dry CH2Cl2 drop-wise
under nitrogen. Then, the reaction mixture was stirred for 5
days at room temperature. After that, the mixture was filtered
and washed with 250 mL of CH2Cl2. The organic phase was
quenched with 10% NaHCO3 and brine, and then dried over
Na2SO4. The solution was concentrated and passed through a
silica-gel column using CH2Cl2 as eluent. After removal of
solvent in vacuo, the polymer was precipitated in cold diethyl
temperature.
Gel-permeation
chromatography
(GPC)
measurements were carried out in Viscotek GPCmax auto
sampler system. Instrument was equipped with a pump
(GPCmax, Viscotek Corp., Houston, TX), light-scattering
detector (λ0 = 670 nm, Model 270 dual detector, Viscotek
Corp.) consisting of two scattering angles: 7° and 90° and the
refractive (RI) index detector (VE 3580, Viscotek Corp.). Both
detectors were calibrated with PS standards in the narrow
molecular weight distribution. Three ViscoGEL GPC columns
(G2000HHR, G3000HHR and G4000HHR) empoyed with THF in
the 1.0 mL min-1 flow rate at 30oC. All data were analyzed
using Viscotek OmniSEC Omni-01 software.
1
ether to yield 26 g (95%). H-NMR (CDCl3, 500 MHz): δ 3.85 –
3.46 (broad), 3.38 (broad), 7.45 (dd, J = 8.5, 2.4 Hz, 1H), 7.52
(d, J = 8.5 Hz, 1H), 7.93 (d, J = 2.4 Hz, 1H).
Synthesis of PPP conjugated polymer (PPP-NH2-g-PEG). The
reaction solution was prepared with 30 mL THF and 20 mL of
2.0 M aqueous K2CO3 solution under nitrogen. Prior use, this
solution was degassed by bubbling nitrogen over a period of
30 min. In the 10 mL of the reaction solution, DBB-MA (0.8 g,
3.0 mmol), DBB-PEG (2.3 g), benzene-1,4-diboronic acid (0.663
g, 4.0 mmol), Pd(PPh3)4) (0.092 g, 0.08 mmol) was dissolved
under nitrogen and stirred at 70oC for 4 days in the Schlenk
tube under vacuum. After stirring, the reaction mixture
extracted with CH2Cl2 and small amount of water. Organic
phase was concentrated and precipitated in excess diethyl
ether to yield 1.85 g, white powder. 1H-NMR (CDCl3, 500 MHz):
δ 2.06 (broad), 3.38 (broad), 3.45-3.8 (broad), 7.92-8.20
(broad), 7.6-7.9 (broad), 7.2-7.6 (broad).
Synthesis of 1,4-dibromo-2-(bromomethyl)benzene (1). 2,5-
Dibromotoluene (2.55 mL, 18.5 mmol), NBS (6.2 g, 24.8 mmol)
and 0.1 g of benzoyl peroxide were added to dry 40 mL of CCl4
under nitrogen atmosphere. The solution was refluxed with
condenser under nitrogen and 200 W light for 4 h. Heating
started from 65 °C and temperature was increased by 10 °C
per hour and in the last one h temperature was kept at 95°C.
After that, the precipitate was filtered and washed with a
supplementary amount of CCl4 and finally with a small quantity
of CH2Cl2. The organic layer was washed with water several
times, and then dried over NaSO4. The solvent was evaporated
in vacuo. Then the remaining crude material was purified by
flash column chromatography (SiO2, Et2O) and recrystallized in
Bioconjugation of FA to PPP-NH2-g-PEG Polymer. FA/PPP-
NH2-g-PEG polymer conjugates were prepared by conventional
EDC/NHS chemistry. FA was conjugated to the polymer
through the reaction between amino group of the polymer and
carboxyl group of FA. Briefly, FA (25 µg/mL) dissolved in PBS
(pH 7.4) reacted with EDC (0.5 M, in 25 mM, pH 5.5 MES
buffer) and NHS (0.125 M, in 25 mM, pH 5.5 MES buffer) for
the activation of the carboxyl group of FA. Then, the activated
FA was reacted with PPP-NH2-g-PEG polymer containing amino
group for 4 h at room temperature. Finally, the conjugates
were washed with PBS three times by using 10 kDa membrane
filter (Sartorius Stedim Biotech).
1
petroleum ether to yield (1) (3.04 g, 50%, white crystal). H-
NMR (CDCl3, 500 MHz): δ 4.54 (s, 2H), 7.30 (dd, J = 8.5, 2.4 Hz,
1H), 7.45 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 2.3 Hz, 1H).
Synthesis of 2-(2,5-dibromobenzyl)isoindoline-1,3-dione (2).
To the solution of
1 (2.9 g, 8.8 mmol) in 10 mL dry DMF,
potassium phthalimide (2.45 g, 13.22 mmol) were added. After
stirring for 12 h at 160 °C in a schlenck tube under nitrogen
atmosphere, the reaction mixture was poured into water. The
precipitate was washed with water, purified by flash column
chromatography (SiO2, CH2Cl2), precipitated in ethanol and
Characterization of the FA/PPP-NH2-g-PEG conjugate. The
structure of polymer can be easily modified with FA due to the
presence of functional amino groups in the structure. To verify
the successful conjugation of polymer and FA, the fluorescence
intensity of polymer was monitored with microplate reader
(Thermo Scientific, Varioskan Flash Multimode Reader, USA)
1
dried under vacuum to yield (2) (2.56 g, 74%, white solid). H-
NMR (CDCl3, 500 MHz): δ 4.94 (s, 2H), 7.26 (dd, J = 5.7, 2.5 Hz,
1H), 7.28 (d, J = 2.3 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.79 (dd, J
= 5.5, 3.0 Hz, 2H), 7.92 (dd, J = 5.5, 3.1 Hz, 2H).
6 | J. Name., 2012, 00, 1-3
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step by step. Also, the conjugation was confirmed FTIR lead new pathways in the bioconjugation field. The novel
analyses. FT-IR spectra were recorded on a PerkinElmer FT-IR targeted fluorescence bio-probe was successfully synthesized
Spectrum One spectrometer.
and applied to both HeLa and A-549 cells for examination of
Cell culture. A549 (human lung cancer) and HeLa (human differences of the cellular affinity between targeted and non-
cervices cancer) cell lines were obtained from ATCC. Cells were targeted samples. All of our results demonstrated that the
cultured on DMEM containing 10% FCS and 1.0% florescent property of synthesized PPP-NH2-g-PEG polymer can
penicillin/streptomycin and were incubated at 37◦C in a 5.0% be appropriate for effective cell imaging application. In
CO2 and 95% air humidified atmosphere.
addition to that, the results showed that PPP-NH2-g-PEG
Cytotoxicity assay. Determination of the dose dependent polymer cannot diffuse into the HeLa and A549 cells when it is
cytotoxicity of the PPP-NH2-g-PEG polymer and prepared in non-conjugated form. The characteristic attributes of the
conjugates was carried out via 3-(4,5-dimethylthiazol-2-yl)-2,5- polymer has shown to be a great model for the targeting
diphenyl tetrazolium bromide (MTT) assay.67 Cells were systems. As a final addition, it was demonstrated that the PPP-
removed from flask to 96-well-tissue plates and were NH2-g-PEG polymer is an excellent candidate for the targeting
cultivated in the wells until reaching confluence. After systems when it is appropriately functionalized.
completion of cultivation, the medium was removed and the
cells were washed with PBS. Following the washing of wells,
Acknowledgements
the cells were treated with the PPP-NH2-g-PEG and FA/PPP-
NH2-g-PEG polymers at 50 µg/mL, 25 µg/mL, 10 µg/mL, 5.0
µg/mL, 1.0 µg/mL concentrations, for 24 h. Then the samples
were removed completely by washing with PBS and 10% MTT
solution was added to the wells. Afterwards, cells were
incubated with MTT for 4 h and 100 µL SDS (1.0 g SDS in 10 mL
0.01 M HCl) was added per well for dissolving formazan which
was occurred inside the cells as a result of MTT treatment.
After 24 h of incubation, the optical densities of each well
were measured by using a spectrophotometric plate reader
(Bio-Tek Instruments, Winooski, VT, USA).
The authors thank to F.B. Barlas (Ege University, Biochemistry
Department) for technical support during the cell culture
studies.
Notes and references
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Cell imaging via fluorescence microscopy. For the purpose of
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imaged via fluorescence microscope (Olympus BX53F)
equipped with a CCD camera(Olympus DP72) by utilizing the
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with high water solubility and stable fluorescence feature may
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